Tumor–Immune Dynamics in Peripheral Blood: Sustained Presence of Stemness-Associated Circulating Tumor Cells During Chemotherapy in Gastric Cancer

Abstract

Background: Circulatin Tumor Cells (CTCs) are promising indicators for gastric cancer tumour dynamics and therapy response. Epithelial and stemness-related gene expression can be detected with great sensitivity using RT-qPCR. This study measured EpCAM (CD326), CD44, CD133, and CK19 in gastric cancer patients' peripheral blood before and throughout chemotherapy and compared them to healthy controls.
Methods: This prospective study had two primary groups: healthy controls (n = 20) and patients with stomach cancer (n = 75). Patient samples were obtained at three intervals: T0 (before to the initial chemotherapy cycle) n=25, T1 (prior to the third cycle) n=25, and T2 (subsequent to the last cycle) n=25. Peripheral blood mononuclear cells were extracted using Ficoll-Paque density gradient centrifugation, subsequently followed by RNA extraction and cDNA synthesis. RT-qPCR quantified the expression of EpCAM, CD44, CD133, and CK19, normalised to GAPDH. The cut-off values for CTC positive were established as the mean plus two standard deviations of healthy controls.
Results: In healthy controls, the mean ± SD expression levels were as follows: EpCAM: 0.15 ± 0.05, CD44: 0.25 ± 0.08, CD133: 0.10 ± 0.04, and CK19: 0.12 ± 0.06. The positivity cut-offs were determined to be 0.25, 0.41, 0.18, and 0.24, respectively. At baseline (T0), patient values were significantly elevated: EpCAM (8.00 ± 1.10), CD44 (6.00 ± 1.48), CD133 (10.00 ± 0.96), and CK19 (12.00 ± 0.153). Notable reductions were recorded at T1 and T2, with the most pronounced decreases for EpCAM and CK19. All markers consistently exceeded their respective cut-off thresholds at T2, indicating the presence of persistent CTCs. The decline of stemness-associated markers CD44 and CD133 was slower, indicating the presence of chemoresistant subpopulations.
Conclusion: The RT-qPCR analysis of EpCAM, CD44, CD133, and CK19 indicates fluctuating circulating tumour cell (CTC) variations throughout treatment in gastric cancer. EpCAM and CK19 diminish swiftly with therapy, signifying a reduction in bulk epithelial circulating tumour cells (CTCs), whereas CD44 and CD133 remain, possibly indicating the presence of residual stem-like CTCs and an increased risk of recurrence. Ongoing molecular positive despite clinical intervention underscores the necessity for integrated therapeutic approaches aimed at chemoresistant circulating tumour cells (CTCs).